PROJECT SUMMARY Bone ailments are costly, painful, and debilitating. Therapies to augment bone repair vary in effectiveness, highlighting the need to develop new treatment approaches. Zebrafish, unlike humans, possess the remarkable capability to completely and robustly regenerate multiple tissues through a process called epimorphic regeneration. A mechanistic understanding of adult zebrafish regeneration could provide clues to unlock this capability in human tissues. Regeneration of the distinct bony rays of zebrafish fins following amputation initiates when mature osteoblasts (Obs) near the site of damage acquire progenitor-like traits by up-regulating key genes, including the transcription factor Runx2, and down-regulating maturation genes, such as the transcription factor sp7/Osterix. The resulting progenitor osteoblasts (pObs) are the singular source of cells from which new bone is derived through their delicately balanced proliferation and re-differentiation until regeneration is complete. pObs are continuously maintained throughout the regeneration process and failure to do so disrupts regeneration. How pObs are maintained in a progenitor state while facing potent differentiation signals remains largely unresolved. Preliminary in vivo and in vitro data suggests that upregulated Ezh2, the histone H3 lysine 27 tri-methylation (H3K27me3) methyltransferase component of the Polycomb Repressive Complex 2 (PRC2), helps sustain pObs. These results support a working hypothesis that Ezh2/H3K27me3 promotes the pOb state through an ?epigenetic barrier? repressing the transcription of genes that drive Ob differentiation. This model will be tested by two Specific Aims: 1) Determine the in vivo contributions of Ezh2 to pOb maintenance in regenerating zebrafish fins and 2) Define how PRC2/Ezh2 maintains pObs. The applicant will use in vitro and in vivo approaches, including CRISPR/Cas9-generated ezh2 mutant zebrafish, inducible transgenic zebrafish and an Ezh2 small molecule inhibitor to perform loss of function and gain of function studies. Further outcomes include a comprehensive description of Ezh2-regulated gene expression programs and H3K27me3 chromatin landscapes in progenitor vs. differentiated osteoblasts. The fellowship period additionally will incorporate mentoring and classroom teaching activities, extensive scientific communication experience and training, and participation in career development workshops.